This invention generally pertains to the field of medicine and pain control. In particular, this invention pertains to the surprising discovery that pia mater cells transformed to secrete beta-endorphin will selectively control chronic pain while not significantly affecting basal nociceptive, acute pain, responses.
This invention is the surprising discovery of a method of using beta endorphin through genetic engineering to treat chronic pain, while at the same time not significantly affecting the ability to react to acutely painful, potentially dangerous, stimuli. As will be explained below, prior to this invention, there was a great amount of academic debate as to whether beta-endorphin can be used to treat or control chronic pain. Thus, the discovery of beta-endorphin""s selective control of chronic pain when secreted by transformed pia mater cells was unpredictable and therapeutically advantageous.
Current analgesic therapies often fall short of therapeutic goals and typically have unacceptable side effects. In many chronic pain syndromes, such as those subsequent to neuropathic injury, pain is not well controlled by any currently available method. Furthermore, most chronic pain treatment regimes affect the patient""s ability to perceive acute pain, thus blunting or abrogating necessary protective basal nociceptive responses.
Exogenous opioids, such as morphine, are commonly used to treat chronic pain. Unfortunately, such drugs are addictive and result in side effects, such as nausea and constipation. Such side effects can be reduced by delivery into the subarachnoid space through intrathecal pumps. However, pumps are expensive and invasive, leading to possible risk of infection. Tolerance and dependence to exogenous opioids is also a severe medical and public health problem.
Endogenous opioids as analgesics for the treatment of acute and chronic pain has also been the subject of many studies. There are numerous endogenous opioids, including, e.g., the beta-endorphin peptide family, enkephalins, dynorphins (see, e.g., Cesselin (1995) Fundam. Clin. Pharmacol. 9:409-433). In particular, investigators have focused on the role and use of beta-endorphins as mediators of the body""s response to pain.
However, studies have generated conflicting theories as to the role beta-endorphin plays in both acute and chronic pain. Thus, to date, the specific role of beta-endorphin in the physiology and neurotransduction of pain has not been determined.
The vast majority of studies implicate beta-endorphin as an endogenous mediator of acute pain and stress. For example, Dionne (1998) Clin. Pharmacol. Ther. 63:694-701, reported that administration of the analgesic ibuprofen suppresses both acute pain and the plasma beta-endorphin levels seen after oral surgery (tooth extraction). In Kamei (1993) Brain Res. 13:619:76-80, intrathecal administration of beta-endorphin had clear anti-nociceptive effects when acute pain was induced (by heat) in rats (tail-flick response). Tseng (1993) Life Sci. 52:PL211-215; also concluded that beta-endorphin induced anti-nociception in a mouse acute pain (tail-flick response) model. Przewlocki (1987) Pol. J. Pharmacol. Pharm. 39:609-621, reported an enhanced release of beta-endorphin in the brain and pituitary in response to acute stimulationxe2x80x94acute pain precipitated a rapid depletion of beta-endorphin in the hypothalamus and midbrain. Puig (1982) Anesthesiology 57:1-4, is one of several studies implicating beta-endorphin as a mediator of acute post-operative pain. Terenius (1982) Acta Anaesthesiol. Scand. Suppl. 74:21-24, concluded that endorphins do have a protective role in acute pain after finding that the better the pain control (by higher levels of pain killer after major surgery), the lower the level of beta-endorphin in the cerebral spinal fluid (CSF). Furthermore, beta-endorphin release is a response associated with acute stress, see, e.g., Guilleman (1977) Science 197:1367-1369; Milan (1981) Mod. Probl. Pharmacopsychiatry 17:49-67; Mueller (1981) Life Sci. 29:1169-1176; Vermes (1981) Neurosci. Lett. 27:89-93; Akil (1985) Science 227:424-426; Millan (1985) Int. Rev. Neurobiol. 26:1-83; Millan (1987) J. Neurosci. 7:77-87.
There is academic debate as to whether or not beta-endorphin can control chronic pain. Conflicting theories have been presented as to the role of beta-endorphin in chronic pain. Some studies suggest that beta-endorphin is not involved in chronic pain. For example, Calvino (1992) Pain 49:27-32, found no beta-endorphin response when chronic pain was maximum (in a rat arthritis animal model). Dehen (1990) Rev. Neurol. (Paris) 146:155-157, measuring concentrations of beta-endorphin in the CSF in painless subjects and patients with chronic pain, found no significant difference between the two groups. France (1991) Psychosomatics 32:72-77, found no change in CSF beta-endorphin concentrations after successful treatment of pain and resolution of depression in patients suffering chronic neuralgic low back pain/sciatica. Salar (1991). Pharmacol. Res. 23:181-186, found no difference in levels of beta-endorphin the CSF of control subjects versus patients with different types of chronic pain (i.e., suffering from deafferentation pain syndromes). Guieu (1992) Pain 48:83-88, found that no increase in beta-endorphin levels occurred concomitantly with pain relief (induced by vibration).
In contrast, there are only a few references suggesting beta-endorphin may be a potential analgesic for chronic pain. One early reference, in 1978 (Almay (1978) Pain 5:153-162), suggested that beta endorphin may be involved in chronic pain relief; however, they found that patients classified as having mainly organic pain syndromes were found to have significantly lower CSF endorphin levels than patients with predominantly psychogenic pain. Lipman (1991) Psycho-pharmacology 102:112-116, found that increased levels of CSF beta-endorphin were correlated with increased pain relief in chronic pain patients. Young (1993) J. Neurosurg. 79:816-825, found a direct relationship between beta-endorphin release in the brain and alleviation of intractable chronic pain. Baxter, et al., U.S. Pat. No. 4,350,764, which teaches the recombinant synthesis of beta endorphin in bacteria, states as background that beta endorphin was known to be useful in the treatment of intractable pain, such as phantom limb pain, Significantly, none of these references administers beta endorphin to treat chronic pain.
Thus, it is clear that, to date, there is no consensus concerning the role of beta-endorphin for controlling chronic pain or its ability to selectively modulate between these distinct pain types. Also, before this invention, there was no effective method of specifically treating chronic pain while at the same time not significantly affecting basal nociceptive (acutely painful) responses. The present invention resolves this controversy by providing a novel means to treat chronic pain. Thus, the ability to preserve sensitivity to acute pain while successfully treating chronic pain is a surprising discovery.
The invention provides a method of treating chronic pain by administering beta-endorphin, wherein basal nociceptive responses are not significantly affected. The beta-endorphin can be administered in the form of an expression cassette, such as an expression vector, for recombinant beta endorphin expression in vivo. The expression vector can be, e.g., any viral vector, such as adenovirus, adeno-associated virus (AAV), lentivirus, or herpes virus, or plasmid, or the like. In a preferred embodiment, the viral vector is a recombinant adenoviral vector or an AAV vector. The beta-endorphin-expressing nucleic acid can be administered by any means, including, e.g., aqueous solutions, lipid-cationic delivery systems, and the like.
In one embodiment, the beta-endorphin-expressing nucleic acid (e.g., an expression cassette, such as a recombinant adenovirus or AAV vector) is administered such that it is transduces a tissue that is anatomically approximate to a nerve, neuron or nerve terminal involved in the transmission or perception of pain, including, e.g., connective tissue (e.g., the epineurium or perineurium), tissue surrounding nerve ganglia, nerve sheathes, nerve linings, or meninges, e.g., the pia mater, or arachnoid or dura membranes; or, cell lining a joint. For example, the recombinant expression systems can be administered to a subarachnoid space or epidural space. In a preferred embodiment, the expression vector is administered into the subarachnoid space. In alternative embodiments, the expression vector is administered in or approximate to joints for, e.g., the treatment of arthritis.
The beta-endorphin-expressing nucleic acid (e.g., recombinant virus or cationic lipid-nucleic acid complex) can be administered in a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be, e.g., an aqueous solution, a lipid delivery system, and the like.
In one embodiment, the beta-endorphin-expressing nucleic acid comprises a nucleic acid encoding a beta-endorphin-secretion signal fusion protein. In alternative embodiments of the method, the nucleic acid insert encoding the beta-endorphin-secretion signal fusion protein is operably linked to a constitutively active promoter to constitutively express the fusion protein, or, it is operably linked to an inducible promoter. The method can further comprising the step of administering a composition capable of inducing the inducible promoter, wherein the fusion protein (e.g., beta-endorphin or a second polypeptide, see below) is expressed upon induction of the promoter.
The recombinant virus concentration in the pharmaceutically acceptable excipient can be between about 103 to about 1018 particles per mL or can be between about 105 to about 1015 particles per mL. In a preferred embodiment, the beta-endorphin-expressing nucleic acid comprises a recombinant virus in a pharmaceutically acceptable excipient at a concentration of between about 106 to about 1013 particles per mL.
Several different expression constructs (e.g., recombinant virii) can be present in the pharmaceutically acceptable excipient. For example, several adenovirus constructs can be co-administered (in a single solution) wherein each constuct contains different forms of beta-endorphin (e.g., various beta-endorphin analogs), different polypeptides (e.g., other pain mediating agents, such as enkaphalin, or markers, such as EGFP, as discussed below), different promoters (e.g., inducible or constitutive), or any combination of polypeptide encoding, transcriptional or translation control or other elements and variations which are apparent to the skilled artisan. In the same manner, different expression vectors can be co-administered, such as, e.g., combinations of adenovirus, AAV, lentivirus, plasmids, and the like.
The virus can be administered into any anatomic area approximate to the spinal chord parenchyma. For example, the virus can be administered into the subarachnoid space or epidurally. After administration the recombinant virus then infects the connective tissue cells surrounding the spinal chord, such as, e.g., the pia mater, arachnoid mater and/or the dura mater. The infected cells express the polypeptide (e.g., the beta-endorphin fusion protein, processed peptide, or fragments of complexes thereof). The fusion protein or a fully processed peptide (e.g., human beta-endorphin peptide) is then secreted. The fusion protein or fragment thereof or a fully processed peptide (e.g., neuroactive peptide) is secreted into the connective tissue interstitial space or cerebral spinal fluid or into (toward) the spinal chord parenchymal tissue in an amount effective to treat chronic pain but not significantly affecting the basal nociceptive responses.
Expression of the in vivo recombinantly generated fusion polypeptide by the transduced connective tissue cells (e.g., pia mater, arachnoid mater, or dura mater cells) results in directed secretion of the fusion protein (or fragment thereof)or a processed peptide into or toward the CSF or the spinal chord parenchymal tissue in an amount effective to treat the chronic pain but not significantly affecting basal nociceptive responses. A similar process occurs when the pharmaceutical composition of the invention is administered into or approximate to a joint space.
In one embodiment of the method, the secretion signal moiety of the fusion protein is a pre-pro sequence of human nerve growth factor (NGF) or any other leader sequence for secretion.
In the methods of the invention the recombinant virus can further comprise a nucleic acid encoding a second polypeptide. The second polypeptide can be the beta-endorphin-secretion signal fusion protein, a partially or fully processed form of beta-endorphin, an analog of beta-endorphin, an enkephalin derived peptide, neurotensin, neuropeptide Y, or fragments or complexes thereof, or enhanced green fluorescence protein (EGFP or eGFP).
In the methods of the invention the nucleic acid encoding the second polypeptide is operatively linked to a promoter different from the promoter to which the beta-endorphin encoding nucleic acid is operatively linked, or, the nucleic acid encoding the second polypeptide is operatively linked to the same promoter the beta-endorphin encoding nucleic acid is operatively linked and the two coding sequences are transcribed as a bicistronic message which is translated to simultaneously produce both polypeptides.
In the methods of the invention the bicistronic message comprises an IRES sequence. The IRES can be the novel IRES of the invention, having a sequence as set forth in SEQ ID NO:4.
The invention also provides a pharmaceutical composition for treating chronic pain, comprising a recombinant virus, such as, e.g., an adenovirus, AAV, lentivirus or herpesvirus, in an excipient pharmaceutically acceptable for in vivo administration, e.g., such as intrathecal or subdural administration. Several different expression constructs (e.g., recombinant virii) can be present in the pharmaceutical compositions of the invention in a variety of combinations as discussed above for pharmaceutically acceptable excipients.
Recombinant virus in the pharmaceutical composition can comprises a nucleic acid insert encoding a beta-endorphin-secretion signal fusion protein. In one embodiment, the recombinant virus (e.g., adenovirus or AAV) concentration in the excipient is between about 103 to about 1018 particles per mL, or between about 105 to about 1015 particles per mL or between about 1 6 to about 1013 particles per mL. The recombinant virus concentration can also be between about 103 to about 1018 infectious units per mL, or between about 105 to about 1015 infectious units per mL or between about 106 to about 1013 infectious units per mL.
When the composition of the invention is administered in an anatomical region approximate to the spinal chord, e.g., intrathecally or subdurally, the recombinant virus (e.g., AAV or adenovirus) infects the meninges, including the pia mater connective tissue cells. The recombinant virus (e.g., AAV or adenovirus), after infecting the connective tissue cells surrounding and/or close to the spinal chord parenchyma (e.g., pia mater), expresses the fusion protein in the infected cell. The infected cell secrets the fusion protein, fragment of the fusion protein or intracellularly processed cleavage product of the fusion protein (e.g., the fully processed peptide) into (or approximate to) spinal chord parenchymal tissue. The fusion protein may be secreted whole but the active moiety is typically the neuroactive peptide or protein that is cleaved (processed intracellularly) from the fusion protein. The leader sequence is usually left behind in the cell. However, the invention includes in vivo synthesis and secretion of all processed, partially processed and unprocessed forms of the polypeptide(s) encoded by the nucleic acids of the pharmaceutial compositions of the invention.
Similarly, recombinant virus, after infecting the connective tissue cells surrounding and/or close joint spaces, is expresses fusion protein in the infected cell and the infected cell secrets the fusion protein or recombinantly generated processed peptide into (or approximate to) the joint space or nerve innervating the joint.
In one embodiment, the nucleic acid insert of the pharmaceutical composition encoding the beta-endorphin-secretion signal fusion protein is operably linked to a constitutively active promoter, wherein the fusion protein is constitutively expressed. In another embodiment, the nucleic acid insert encoding the beta-endorphin-secretion signal fusion protein is operably linked to an inducible promoter, wherein the fusion protein is expressed upon induction of the inducible promoter. The secretion signal moiety of the fusion protein can be a pre-pro sequence of human nerve growth factor (NGF) or any other leader sequence for secretion (see, e.g., Ritty (1999) J. Biol. Chem. 274(13):8933-40; Sasada (1988) Cell Struct. Funct. 13(2):129-41).
The pharmaceutically acceptable excipient can be an aqueous solution, a lipid solution, a mixture thereof, and the like. In the pharmaceutical composition of the invention the pharmaceutically acceptable excipient can be an aqueous solution or a lipid based solution (e.g., including liposomes or cationic or anionic lipid complexes).
In the pharmaceutical composition of the invention the recombinant virus can further comprise a nucleic acid encoding a second polypeptide. The second polypeptide can be any polypeptide or peptide that mediates pain or nerve or neuron function or their supporting tissues, e.g., beta-endorphin-secretion signal fusion protein, a human beta endorphin, an analog of beta-endorphin, an enkephalin derived peptide neurotensin, neuropeptide Y, or fragments or complexes of any of these proteins.
The second polypeptide can also be any marker or identifier (e.g., immunoreactive, with predefined epitope) protein, e.g., any intrinsically fluorescent protein, e.g., enhanced green fluorescence protein (eGFP) and the like.
In the pharmaceutical composition of the invention the nucleic acid encoding the second polypeptide is operatively linked to a promoter different from the promoter to which the beta-endorphin encoding nucleic acid is operatively linked, or, the nucleic acid encoding the second polypeptide is operatively linked to the same promoter the beta-endorphin encoding nucleic acid is operatively linked with and the two coding sequences are transcribed as a bicistronic message which is translated to produce both polypeptides. In the pharmaceutical composition the bicistronic message can comprises an IRES sequence. The IRES can be the novel IRES of the invention having a sequence as set forth in SEQ ID NO:4.
The invention also provides a nucleic acid sequence comprising a sequence as set forth in SEQ ID NO:4, or a nucleic acid sequence consisting essentially of the sequence as set forth in SEQ ID NO:4, or, a nucleic acid sequence having a sequence as set forth in SEQ ID NO:4.
A further understanding of the nature and advantages of the present invention is realized by reference to the remaining portions of the specification, the figures and claims.
All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.